This invention relates to mutations in nucleic acid molecules encoding the protein 11-cis retinol dehydrogenase, or xe2x80x9cRDH5,xe2x80x9d and the resulting mutated protein. These mutations are implicated in ocular disorders, such as fundus albipunctatus. The diagnostic and therapeutic ramifications of these mutations are also discussed and are features of the invention.
Retinoids (vitamin A-derivatives) have important physiological functions in a variety of biological processes. During embryonic growth and development, as well as during growth and differentiation of adult organisms, retinoids act as hormones and participate in the regulation of gene expression in a number of cell types. See Lied et al. Trends Genet., 17:427-433 (1992). It is believed that these effects are medicated through two classes of nuclear ligand-controlled transcription factors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs), Benbrook et al., Nature, 333:669-672 (1988); Brand et al., Nature, 332:850-853 (1988); Giguere et al., Nature, 330:624-629 (1987); Mangelsdorf et al., Nature, 345:224-229 (1990); Mangelsdorf, et al. Genes Dev. 6:329-344 (1992); Petkovich et al. Nature 330:440-450 (1987); and Zelent et al., Nature 339:714-717 (1989).
Apart from their function as hormones in cellular growth and differentiation, retinoids are also involved in the visual process, as the stereo isomer 11-cis retinaldehyde is the chromophore of the visual pigments. See, e.g. Bridges, The Retinoids, Vol. 2, pp 125-176, Academic Press, Orlando, Fla., (1984).
Under normal physiological conditions most cells, both ocular and non-ocular, obtain all-trans retinol as their major source of retinoids. Despite the many different metabolic events taking place in different tissues, it is known that a common extracellular transport machinery for retinol has evolved. Specifically, in plasma, retinol is transported by plasma retinol binding protein (RBP). See Goodman et al., The Retinoids, Academic Press, Orlando Fla., Volume 2, pp. 41-88 (1984). The active derivatives of retinol, retinoic acid in non-ocular tissues and mostly 11-cis retinaldehyde for ocular tissues, are then generated by cellular conversion using specific mechanisms. To date, none of these mechanisms have been fully defined at the molecular level and several of the enzymes involved have only been identified by enzymatic activities. See Lion et al., Biochem. Biophys. Acta. 384:283-292 (1975); Zimmermann et al., Exp. Eye Res. 21:325-332 (1975); Zimmerman, Exp. Eye Res. 23:159-164 (1976) and Posch et al., Biochemistry 30:6224-6230 (1991).
Polarized retinal pigment epithelial cells (RPE) are unique with regard to retinoid uptake since all-trans retinol enters these cells via two different mechanisms. Retinol accumulated from RBP is taken up through the basolateral plasma membrane, while all-trans retinol, presumably taken up from the interstitial retinol-binding protein (IRBP) following bleaching of the visual pigments, may enter through the apical plasma membrane. See Bok et al., Exp. Eye Res. 22:395-402 (1976); Alderetal., Biochem. Biophys. Res. Commun. 108:1601-1608(1982); Lai et al., Nature 298:848-849 (1982); and Inu et al., Vision Res. 22:1457-1468 (1982).
The transfer of retinol from RBP to cells is a subject under investigation. In a number of cell types, including RPE, specific membrane receptors for RBP have been identified, which is consistent with a receptor-mediated uptake mechanism for retinol. For example, isolated retinol binding protein receptors, nucleic acid molecule coding for these receptors and antibodies binding to the receptor are known. . These teachings relate to the first of the two mechanisms. See Bavik et al., J. Biol. Chem. 266:14978-14985 (1991); Bavik, et al. J. Biol. Chem. 267:23035-23042 1992; Bavik et al., J. Biol. Chem. 267:20540-20546 (1993); and U.S. Pat. Nos. 5,573,939 and 5,679,772, all of which are incorporated by reference. See also Heller, J. Biol. Chem. 250:3613-3619 (1975); and Bok et al., Exp. Eye Res. 22:395-402 (1976).
Retinol uptake on the apical side of the RPE for the regeneration of 11-cis retinaldehyde (xe2x80x9c11-cis retinalxe2x80x9d hereafter) is less well characterized. However, regardless of the origin of all-trans retinol, the synthesis and apical secretion of 11-cis retinal seems to be the major pathway for accumulated retinol in the RPE. At present, it is not known whether similar mechanisms are used with regard to cellular retinol uptake through the basolateral and the apical plasma membranes. However, available data show that functional receptors for RBP are exclusively expressed on the basolateral plasma membrane of RPE-cells. Bok et al., Exp. Eye Res. 22:395-402 (1976).
It is also known that retinal pigment epithelial cells (RPE) express a 63 kDa protein (p63). It has also been shown by chemical cross-linking that this protein may be part of an oligomeric protein complex which functions as a membrane receptor for plasma retinol-binding protein (RBP) in RPE-cells, or a component of the retinoid uptake machinery in RPE cells. See Bavik et al., J. Biol. Chem. 266:14978-14875 (1991); Bavik et al., J. Biol, Chem. 267:23035-23042 (1992), and U.S. Pat. Nos. 5,573,939 and 5,679,772 The p63 protein has been isolated and the corresponding cDNA cloned. See Bavik et al., J. Biol. Chem. 267:20540-20546 (1993)and the ""939 and ""772 patents referred to supra. All of these references are incorporated by reference.
11-cis retinal, referred to supra is important in vision, because it is the light sensing chromophore found in cone opsins and rod opsins (i.e., xe2x80x9crhodopsinxe2x80x9d), in both cone and rod photoreceptor cells. Deficiencies in vitamin A result in reduction in concentrations of rhodopsin in the retina, which is followed by night blindness. In turn, if night blindness is left untreated it is followed by degeneration of rod photoreceptors, and then cone photoreceptors. In fact, vitamin A supplementation has been reported to slow the course of retinal degeneration in diseases such as retinitis pigmentosa (Berson et al, Arch. Ophthalmol111:761-772(1993), and to reverse the night blindness found least temporarily. See Jaconson, et al, Nature Genet. 11:27-32(1995).
Deficiencies in vitamin A can be attributed to one or more causes, including poor diet, or a deficiency in one or more of the proteins involved in transport of vitamin A through the bloodstream. See, e.g., Wetterau, et al., Science 258:999-1001 (1992), and Narcisi, et al., Am. J. Hum. Genet 57:1298-1310 (1995), discussing an inherited deficiency in microsomal triglyceride transfer protein, and Seeliger, et al., Invest. Ophthal Vis. Sci. 40:3-11 (1999), discussing an inherited deficiency in serum retinol binding protein.
Physiological abnormalities and visual symptoms also arise from defects in the storage or metabolism of vitamin A within the retina. With respect to the storage of the vitamin, a number of proteins are thought to bind 11-cis and all-trans vitamin A alcohols and aldehydes in the retina and the retinal pigment epithelium. These proteins include xe2x80x9cCRALBPxe2x80x9d, or cellular relinaldehyde binding protein, xe2x80x9cIRBPxe2x80x9d, or xe2x80x9cinter-photoreceptor retinoid binding proteinxe2x80x9d, and xe2x80x9cCRBPxe2x80x9d, or cellular retinol-binding protein. CRALBP and IRBP are known to be essential to photoreceptor physiology, since null mutations in the genes encoding these proteins cause photoreceptor degeneration in mammals. See Mau, et al., Nature Genet 17:198-200 (1997); Morimura, et al., Invest. Ophthalmal. Vis. Sci. 40:1000-1004 (1999); Burstedt, et al., Invest. Ophthal. Vis. Sci. 40:995-1000 (1999); Liou, et al., J. Neurasci 18:4511-4520 (1998).
In contrast to the understanding of the pathways and mechanisms discussed supra, abnormalities in the metabolic pathways which convert all-trans retinol from the bloodstream into 11-cis retinal, and that reconvert the all-trans retinal produced after cone and rod photopigments absorb photons of light back to 11-cis retinol are not well understood. Various enzymes are involved in this pathway, and are found in photoreceptor cells, as well as neighboring retinal pigment epithelium, and Muller cells of the retina. Several of these enzymes have been purified only recently. For example, see Simon et al., J. Biol. Chem 270:1107-1112 (1995), and U.S. Pat. No. 5,731,195, both of which are incorporated by reference for teachings relating to purified 11-cis retinal dehydrogenase, and molecules encoding this enzyme, Haeseleer, et al., J. Biol. Chem 273:21790-21799 (1998), for teachings related to all-trans retinal oxido reductase, and Ruiz, et al., J. Biol. Chem 274:3834-3841 (1999), for teachings relating to
It has now been found that ocular disorders are associated with mutations in the nucleic acid molecules encoding 11-cis retinal dehydrogenase (RDH5), and the resulting mutated proteins. These are features of the invention which are set out in the disclosure which follows.